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Donepezil {(RS)-2-[(1-benzyl-4-piperidyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one} is a reversible acetylcholinesterase inhibitor and used for treatment of patients with AD (Alzheimer's disease). Recent studies showed that treatment with donepezil reduced production of inflammatory cytokines in PBMCs (peripheral blood mononuclear cells). It was also reported that muscle-derived inflammatory cytokines play a critical role in neovascularization in a hindlimb ischaemia model. We sought to determine whether donepezil affects angiogenesis. A hindlimb ischaemia model was created by unilateral femoral artery ligation. Blood flow recovery examined by laser Doppler perfusion imaging and capillary density by immunohistochemical staining of CD31-positive cells in the ischaemic hindlimb were significantly decreased in donepezil- and physostigmine-treated mice compared with control mice after 2 weeks. Donepezil reduced expression of IL (interleukin)-1β and VEGF (vascular endothelial growth factor) in the ischaemic hindlimb. Intramuscular injections of IL-1β to the ischaemic hindlimb reversed the donepezil-induced VEGF down-regulation and the anti-angiogenic effect. Hypoxia induced IL-1β expression in C2C12 myoblast cells, which was inhibited by pre-incubation with ACh (acetylcholine) or LY294002, a PI3K (phosphoinositide 3-kinase) inhibitor. Donepezil inhibited phosphorylation of Akt [also known as PKB (protein kinase B)], a downstream kinase of PI3K, in the ischaemic hindlimb. These findings suggest that cholinergic stimulation by acetylcholinesterase inhibitors suppresses angiogenesis through inhibition of PI3K-mediated IL-1β induction, which is followed by reduction of VEGF expression. Acetylcholinesterase inhibitor may be a novel anti-angiogenic therapy.

Inhibition of prolyl hydroxylase domain-containing protein (PHD) by hypoxia stabilizes hypoxia-inducible factor 1 and increases the expression of target genes, such as vascular endothelial growth factor. Although the systemic renin-angiotensin system is activated by hypoxia, the role of PHD in the regulation of the renin-angiotensin system remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin II type 1 receptor (AT 1 R). Hypoxia, cobalt chloride, and dimethyloxalylglycine, all known to inhibit PHD, reduced AT 1 R expression in vascular smooth muscle cells. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT 1 R expression. Cobalt chloride diminished angiotensin II–induced extracellular signal–regulated kinase phosphorylation. Cobalt chloride decreased AT 1 R mRNA through transcriptional and posttranscriptional mechanisms. Oral administration of cobalt chloride (14 mg/kg per day) to C57BL/6J mice receiving angiotensin II infusion (490 ng/kg per minute) for 4 weeks significantly attenuated perivascular fibrosis of the coronary arteries without affecting blood pressure level. These data suggest that PHD inhibition may be beneficial for the treatment of cardiovascular diseases by inhibiting renin-angiotensin system via AT 1 R downregulation.

Objective— Prolyl hydroxylase domain-containing proteins (PHDs) play pivotal roles in oxygen-sensing system through the regulation of α-subunit of hypoxia-inducible factor (HIF), a key transcription factor governing a large set of gene expression to adapt hypoxia. Although tissue hypoxia plays an essential role in maintaining inflammation, the role of PHDs in the inflammatory responses has not been clearly determined. Here, we investigated the role of PHDs in lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF-α) induction in macrophages. Methods and Results— Northern blot analysis and ELISA revealed that LPS-induced TNF-α upregulation was strongly suppressed by PHD inhibitors, dimethyloxallyl glycine (DMOG), and TM6008 in RAW264.7 macrophages. DMOG suppressed LPS-induced TNF-α upregulation in HIF-1α–depleted cells and HIF-1α overexpression failed to suppress the induction of TNF-α. DMOG rather suppressed LPS-induced NF-κB transcriptional activity. Downregulation of Phd1 or Phd2 mRNA by RNA interference partially attenuated LPS-induced TNF-α induction. DMOG also inhibited LPS-induced TNF-α production in peritoneal macrophages as well as human macrophages. Conclusions— PHD inhibition by DMOG or RNA interference inhibited LPS-induced TNF-α upregulation in macrophages possibly through NF-κB inhibition, which is independent of HIF-1α accumulation. This study suggests that PHDs are positive regulators of LPS-induced inflammatory process, and therefore inhibition of PHD may be a novel strategy for the treatment of inflammatory diseases.